Reprogramming of cells by nuclear transfer (Wakayama et al., 1998; Wilmut et al., 1997) and cell fusion (Cowan et al., 2005; Tada et al., 2001) allows for the re-establishment of a pluripotent state in a somatic nucleus (Hochedlinger and Jaenisch, 2006). While the molecular mechanisms of nuclear reprogramming remain elusive, cell fusion experiments have implied that reprogramming factors can be identified in ES cells and be used to directly induce reprogramming in somatic cells. Indeed, a rational approach recently led to the identification of four transcription factors whose expression enabled the induction of a pluripotent state in adult fibroblasts (Takahashi and Yamanaka, 2006). Yamanaka and colleagues demonstrated that retroviral expression of the transcription factors Oct4, Sox2, c-Myc, and Klf4, combined with genetic selection for Fbx15 expression, gives rise to induced pluripotent stem (iPS) cells directly from fibroblast cultures. Fbx15-selected iPS cells contributed to diverse tissues in mid-gestation embryos, however, these embryos succumbed at mid-gestation, indicating a restricted developmental potential of iPS cells compared with ES cells. Consistent with this observation, only part of the ES cell transcriptome was expressed in iPS cells, and methylation analyses of the chromatin state of the Oct4 and Nanog promoters demonstrated an epigenetic pattern that was intermediate between that of fibroblasts and ES cells.
These observations raised three fundamental questions about the molecular and functional nature of directly reprogrammed cells: (i) can selection for a gene that is essential for the ES cell state generate pluripotent cells that are more similar to ES cells than the previously described Fbx15-selected iPS cells; (ii) does the pluripotent state of iPS cells depend on continuous expression of exogenous factors; and (iii) does transcription factor-induced reprogramming reset the epigenetic landscape of a fibroblast genome into that of a pluripotent cell.
Ectopic expression of the transcription factors Oct4, Sox2, cMyc, and Klf4, as well as variants of this factor combination, are sufficient to confer a pluripotent state upon several differentiated cell types, generating induced pluripotent stem cells (iPSCs) (Takahashi, K., and Yamanaka, S. (2006) Cell 126, 663-676; Takahashi, K., et al., (2007) Cell 131, 861-872; Yu, J., et al. (2007) Science 318, 1917-1920; Feng, B., et al., (2009) Nat Cell Biol 11, 197-203; Eminli, S., et al., (2008); Hanna, J., (2008) Cell 133, 250-264; Stadtfeld, M., et al., (2008) Curr Biol 18, 890-894; Aoi, T., et al., (2008) Science 321, 699-702). The derivation of iPSCs is a highly inefficient process with the underlying mechanisms largely unknown. This low efficiency argues for the existence of additional cooperative factors, whose identification is critical for understanding the process of reprogramming. Further, the therapeutic use of iPSCs relies on developing efficient non-genetic means of factor delivery, and while a handful of compounds that replace individual factors have been identified, their use yields a further reduction to the already low efficiency of reprogramming (Huangfu, D., et al., (2008) Nat. Biotechnol.; Shi, Y., (2008) Cell Stem Cell 3, 568-574; Marson, A., (2008) Cell Stem Cell 3, 132-135). Thus, the identification of compounds that enhance rather than solely replace the function of the reprogramming factors will be of great use.